AT III is a serine protease inhibitor present in blood in a large amount (from 20 to 30 mg/dl) and is known as a coagulation inhibitor. As described hereinafter, the blood coagulation reaction includes an intrinsic coagulation reaction pathway and an extrinsic coagulation reaction pathway. Various coagulation factors take part in respective pathways and AT III inhibits most of the activated coagulation factors. Particularly, as the action of AT III, the inhibition of thrombin, which is activated factor II (IIa), and activated factor X (Xa) is considered to be of importance. And, since the reaction of coagulation factors is inhibited more effectively at an early stage in the blood coagulation system, AT III is considered to play a particularly important role in the inhibition of factor Xa which takes part in a stage earlier than factor IIa.
Clinically, attention is directed to AT III in AT III production depressed conditions such as hepatocirrhosis and undernutrition, AT III consuming conditions such as disseminated intravascular coagulation (DIC) and other progressive coagulation conditions, conditions wherein AT III is lost into urine such as nephrosis syndrome, congenital AT III deficiency disease and the like. Therefore, for diagnosis of these conditions, it has been requested to establish a precise, rapid and easy method for determination of biological activity of AT III.
Hitherto, as a method for determination of biological activity of AT III, there have been known a method using a synthetic substrate and a method utilizing coagulation activity. Among them, the method using synthetic substrate is carried out by adding a synthetic substrate and an excess amount of factor IIa to a specimen plasma and measuring anti-IIa activity of AT III in the specimen. This method is excellent because the determination can be carried out within a short period of time. However, the method has many problems. for example, the method has different specificity depending upon the kind of synthetic substrates and a synthetic substrate is very expensive. Further, factor IIa to be used is less stable and, since IIa factor is apt to be adsorbed by glass, a plastic cell should be used in the measurement. Furthermore, since the method requires measuring apparatuses such as a spectrophotometer and a fluorophotometer, the economical burden becomes large.
On the other hand, in the coagulation method, a concentration of AT III is determined by subjecting a specimen plasma to heat treatment to remove fibrinogen, adding an excess amount of factor IIa and measuring a coagulation time due to conversion of fibrinogen to fibrin to obtain the anti-IIa activity of AT III in the specimen. However, these operations of this method are complicated and the determination takes a long period of time, for example, not less than one hour.